Expansion of the genetic code by incorporation of nonstandard amino acids (nsAAs) into proteins has emerged as a powerful approach for template-based incorporation of over 100 nsAAs containing diverse chemical groups, including post-translational modifications, photocaged amino acids, bioorthogonal reactive groups, and spectroscopic labels (Liu, et al., Annu Rev Biochem, 79:413-44 (2010); Johnson, et al., Curr Opin Chem Biol, 14:774-80 (2010); O'Donoghue, et al., Nat Chem Biol, 9:594-8 (2013); Chin, et al., Annu Rev Biochem, (2014); Seitchik, et al., J Am Chem Soc, 134:2898-901 (2012)). For example, site-specific incorporation of nsAAs at a single position enables engineering of protein-drug conjugates (Tian, et al., Proc Natl Acad Sci USA, 111:1766-71 (2014), cross-linking proteins (Furman, et al., J Am Chem Soc, 136:8411-7 (2014), and enzymes with altered or improved function (Kang, et al., Chembiochem, 15:822-5 (2014); Wang, et al., Angew Chem Int Ed Engl, 51:10132-5 (2012)). Multi-site nsAA incorporation can further expand the function and properties of proteins and biomaterials by enabling synthesis of polypeptide polymers with programmable combinations of natural and nonstandard amino acids. However, multi-site nsAA incorporation has so far been limited by inefficiencies associated with the translation machinery and the cellular hosts in which the recombinant proteins are produced (Li, et al., Chembiochem (2014)).
Currently, there are two common approaches to recombinant protein expression with nsAAs. The first approach introduces an nsAA by complete amino acid replacement wherein a natural amino acid is substituted for a close synthetic analog (i.e., the nsAA) in an auxotrophic strain (Dougherty, et al., Macromolecules, 26:1779-1781 (1993)). This approach has been utilized extensively to tag, identify, and study newly synthesized proteomes in a variety of cell types (Dieterich, et al., Proc Natl Acad Sci USA, 103:9482-7 (2006); Yuet, et al., Ann Biomed Eng, 42:299-311 (2014)). In addition, multi-site incorporation of nsAAs using this method has generated biomaterials with improved stability (Tang, et al., Angew Chem Int Ed Engl, 40:1494-1496 (2001); Nishi, et al., Biochemistry, 44:6034-42 (2005)) biopolymers containing conductive chemical groups (Kothakota, et al., Journal of the American Chemical Society, 117:536-537 (1995)), and facilitated characterization of structural proteins (Bae, et al., J Mol Biol, 309:925-36 (2001)). However, complete amino acid replacement has drawbacks that limit its application. First, the chemical diversity introduced via nsAAs in this procedure is limited since the nsAA must be a close analog of the natural amino acid it replaces, a constraint that can be partially alleviated by mutations to the native translation machinery (Kirshenbaum, et al., Chembiochem, 3:235-7 (2002)). Second, the substitution of an nsAA excludes the use of the eliminated amino acid in the recombinant protein (Link, et al., Curr Opin Biotechnol, 14:603-9 (2003)) and replaces it in the entire proteome, causing growth defects which can reduce protein yields.
Alternatively, nsAAs can be incorporated via codon reassignment or frameshift codons using orthogonal translation systems (OTSs) consisting of an aminoacyl tRNA synthetases (“AARS”) that is only able to charge a cognate tRNA, which is not aminoacylated by endogenous AARSs (Liu, et al., Annu Rev Biochem, 79:413-44 (2010); Chin, et al., Annu Rev Biochem, (2014)). Typically, a TAG stop codon (transcribed to UAG during mRNA synthesis) is assigned to the nsAA and the orthogonal tRNA anticodon is mutated to CUA for site-specific nsAA incorporation. Extensive work has demonstrated that AARS:tRNA pairs from divergent organisms such as Methanocaldococcus jannaschii and Methanosarcina species can be imported to bacterial hosts and used to generate OTSs for nsAA incorporation by plasmid library mutagenesis and iterative positive/negative selections (Liu, et al., Annu Rev Biochem, 79:413-44 (2010); Park, et al., Science, 333:1151-4 (2011); Umehara, et al., FEBS Lett, 586:729-33 (2012)). This approach enabled genetic code expansion to a wide variety of nsAAs (Liu, et al., Annu Rev Biochem, 79:413-44 (2010); Young, et al., Biochemistry, 50:1894-900 (2011)). However, several challenges have limited the impact of this technology to expression of proteins containing nsAAs incorporated into a single or few instances within a polypeptide chain (O'Donoghue, et al., Nat Chem Biol, 9:594-8 (2013); Li, et al., Chembiochem (2014)).
The first challenge for multi-site nsAA incorporation using codon-reassignment is competition between the orthogonal nsAA-tRNACUA and essential translation machinery for the UAG codon (e.g., release factor 1, RF1), that reduces full-length protein production and limits the number of nsAAs that can be incorporated into a single protein (Johnson, et al., Nat Chem Biol, 7:779-86 (2011); Lajoie, et al., Science, 342: 357-60 (2013); Heinemann, et al., FEBS Lett, 586:3716-22 (2012); Mukai, et al., Nucleic Acids Res, 38:8188-95 (2010)). To address this, a genomically recoded organism (GRO) was created that recoded all instances of the TAG codon to the synonymous TAA codon in E. coli (Lajoie, et al., Science, 342: 357-60 (2013). This GRO permitted the deletion of RF1, and hence, elimination of translational termination at UAG codons. In this organism, TAG has been transformed from a nonsense codon (terminates translation) to a sense codon (incorporates amino acid of choice), provided the appropriate translation machinery is present (Lajoie, et al., Science, 342: 357-60 (2013); Isaacs, et al., Science, 333:348-53 (2011)).
Nevertheless, a second challenge to multi-site nsAA incorporation via codon reassignment is that the evolved AARSs show ˜100- to 1000-fold reduced enzyme activity (O'Donoghue, et al., Nat Chem Biol, 9:594-8 (2013); Umehara, et al., FEBS Lett, 586:729-33 (2012)) compared with native translation machinery. This results in inefficient nsAA acylation by AARSs (Umehara, et al., FEBS Lett, 586:729-33 (2012); Wiltschi, et al., Yeast, 25:775-86 (2008); Nehring, et al., PLoS One, 7:e31992 (2012)) and subsequent low levels of nsAA-tRNA, reducing protein yields (Lajoie, et al., Science, 342: 357-60 (2013); Zaher, et al., Cell, 136:746-62 (2009); Odoi, et al., PLoS One, 8:e57035 (2013)). This effect is magnified when more than a single nsAA is encoded per protein (Johnson, et al., Nat Chem Biol, 7:779-86 (2011)). It is believed that current approaches rely on multi-copy plasmids for OTS overexpression (i.e., AARS and tRNA overexpression) to overcome enzyme inefficiency, which masks differences between modestly- and highly-active AARSs and prevents the identification of more efficient variants capable of multi-site nsAA incorporation. Therefore, there remains a need for improved compositions and methods for making polypeptides with multi-site nsAA incorporation.
It is an object of the invention to provide improved genomically recoded organisms (GRO) capable of multi-site nsAA incorporation.
It is another object of the invention to provide improved variant aminoacyl tRNA synthetases (AARS) and tRNA that can charge tRNA with a nonstandard amino acid.
It is another object of the invention to provide methods of making improved genomically recoded organism (GRO), aminoacyl tRNA synthetases (AARS), and tRNA.
It is another object of the invention to provide methods of making polypeptides including one or more non-standard amino acids, preferably two or more iterations of the non-standard amino acid or amino acids with a high purity and yield.
It is another object of the invention to provide polypeptides including one or more non-standard amino acids, preferably two or more iterations of the non-standard amino acid or amino acids.